The fusion of mouse myeloma cells to spleen cells was demonstrated by Kohler and Milstein (Nature 256, 495-497, 1975) allowing the generation of continuous cell lines making homogeneous (so-called "monoclonal") antibody. Subsequently, much effort has been directed toward the production of various hybrid cells (called hybridomas) and to uses of antibodies made by these hybrid cells. While the general technique is well understood conceptually, difficulties may be encountered in individual cases. Thus, the technique must be varied to meet the requirements of each specific case. There is no assurance, prior to attempting to prepare a given hybridoma, that the desired hybridoma will be obtained. There is no assurance that it will produce antibody, or that the antibody so produced will have the desired specificity.
Few instances of production of monoclonal antibody to bacterial antigens have been reported. The reported hybridomas produce antibody to non-peptidoglycan antigens unique to a single bacterial genus, species, type or strain, not to common antigens shared by all or nearly all bacteria. Ruch, F. E., Jr. and L. Smith, J.Clin.Microbiol. 16, 145-152 (1982); Nahm, M. H., B. L. Clevinger and J. M. Davie, J.Immunol. 129, 1513-1518 (1982).
Polyclonal antisera to peptidoglycan have been reported. Zeiger, A. R., C. V. Tuazon, and J. N. Sheagren, Inf.&Imm. 33, 795-800 (1981); Schleifer, K. H., and P. H. Seidl, Eur.J.Biochem. 43, 509-519 (1974); Rolicka, M. and J. T. Park, J.Immunol. 103, 196-203 (1969).
True bacteria, referred to as Eubacteria, are considered to be among the group of microorganisms called prokaryotes. Basically, prokaryotes are organisms of small overall dimensions that possess a relatively simple and primitive cellular structure. Unlike eukaryotic cells (which include mammalian cells, algae, fungi, and protozoa), prokaryotic cells lack a nuclear membrane so that the single circular prokaryote chromosome is bathed in cytoplasm. Prokaryotic cells also lack true intracellular organelles such as mitochondria and lysosomes which are enclosed by membranes.
In addition to Eubacteria, prokaryotes include a group of organisms called Archaebacteria (primitive bacteria). Kandler, O., Zbl.Bakt.Hyg., I.Abt.Orig. C 3, 149-160 (1982). This last group does not contain any organisms of known importance to human disease. It includes some extreme halophiles and thermoacidophiles that can be clearly distinquished from bacteria by differences in the structure of their cell surface.
Eubacteria include Mycoplasmatales, which differ from other prokaryotic cells in their lack of a peptidoglycan-containing cell wall. "Peptidoglycan" is also known as "murein" or "mucopeptide."
In the absense of any stain, Eubacteria are very difficult to see under a light microscope. The most commonly used stain, the gram stain, divides Eubacteria into two structurally, chemically, physiologically and medically important groups. "Gram-positive" species retain primary stain (crystal violet), after treatment with a mordant (iodine) and a decolorization procedure using ethanol or acetone. They have a relatively thick, polysaccharide-containing cell wall. "Gram-negative" species lose the primary stain-iodine complex during the decolorizing step. So that all cells are easily visible, a pink-colored counterstain is used. They possess a cell wall containing a large amount of lipid, particularly in the portion of the cell wall commonly called the "outer membrane".
It is understood that all Eubacteria (true bacteria) with the exception of the Mycoplasmatales, i.e. mycroplasma and acholeplasma, have been shown to possess peptidoglycan, a unique cell wall polymer that contains a novel amino sugar in its structure, muramic acid. The peptidoglycan polymer is essential for the growth and survival of bacteria in most environments, and peptidoglycan assembly on the exterior of the cytoplasmic membrane can be selectively interrupted by the appropriate application of certain chemotherapeutic agents.
Peptidoglycan is not present in eukaryotic cells. Such cells include mammalian, plant, protozoan and fungal cells. Peptidoglycan is not present in Archaebacteria or viruses.
Eubacteria, contain the major bacterial pathogens of man and animals. Eubacteria include the following genera: Escherichia, Pseudomonas, Proteus, Micrococcus, Acinetobacter, Klebsiella, Legionella, Neisseria, Bordetella, Vibrio, Staphylococcus, Lactobaccilus, Streptococcus, Bacillus, Corynebacteria, Mycobacteria, Clostridium, and others. Kandler, O., Zbl. Bakt.Hyg., I.Abt.Orig. C3, 149-160 (1982).
Peptidoglycan consists of glycan chains composed of N-acetylglucosamine and N-acetylmuramic acid linked by .beta.-1-4-glycosidic bonds. Muramic acid is a nine-carbon amino sugar that is present only in Eubacteria and can be considered to be N-acetylglucosamine with a lactyl side chain on carbon 3. The peptide side chains of peptidoglycans are covalently (amide) linked to the carboxyl of the lactyl moiety of the muramic acid residues. A unique feature of this macromolecule that contributes to its insolubility, strength, and probably also its shape, lies in the peptide bond between the peptide side chains, resulting in a cross-linked, two- or three-dimensional structure. These peptide cross-links differentiate peptidoglycan from cellulose of plants and chitin of fungi and crustaceans.
A further peculiarity of bacterial cell wall peptidoglycans lies in the chemistry of the peptide side chains. Common features and differences in amino acid composition of these peptides can be illustrated as follows: The amino acid amide linked to N-acetylmuramic acid is almost always L-alanine. The second amino acid in the sequence is usually a D-amino acid, most frequently D-glutamate (or D-glutamine). The third amino acid is linked to this D-amino acid, not to the conventional .alpha.-carboxyl group found in proteins but to the other (.gamma.) carboxyl group, resulting in the presence of the entire carbon skeleton of the second amino acid in the chain. This third amino acid is usually an L-(di)amino acid such as L-lysine or mesodiaminopimelic acid (DAP). DAP is another compound found only in eubacterial cells. When examined, it is the L-isomeric center of DAP (or other diamino acid) that is peptide linked to the second amino acid. The fourth amino acid is almost always D-alanine. Thus, the usual peptide side chain has an L-D-L-D sequence, different from the all L-amino acid sequence of proteins, and resistant to most proteinases, including the enzymes present in the digestive tract.
Cross-linking of the peptide side chains occurs usually between the second amino group of the diamino acid in position 3 of a peptide on one glycan strand and the carboxyl group of terminal D-alanine on a second glycan strand. In some species, this is a direct linkage from the .epsilon.-amino group of DAP or L-lysine to the carboxyl of D-alanine. In other species, one or more amino acids may be present in the bridge between the .epsilon.-amino group of L-lysine and D-alanine.
The chemical composition and structure of the peptidoglycan of an individual bacterial species is known to remain constant under a variety of environmental conditions. Ghuysen, J. M. and G. D. Shockman in Bacterial Membranes and Walls, pp. 37-130 (1973); Rogers, H. J. et al., Microbial Cell Walls and Membranes (1980). This consistency has led to the suggestion that peptidoglycan composition and chemical structure is useful for taxonomy. Kandler, O., Zbl. Bakt.Hyg., I.Abt.Orig. C 3, 149-160 (1982); Schleifen, K. H. and Kandler, O., Bacteriol. Rev. 36, 407-477 (1972).
Common features of peptidoglycan chemistry are believed responsible for the cross-reactivity of single monoclonal antibodies of the present invention to peptidoglycans from widely different bacterial species. Differences in peptidoglycan chemistry are believed responsible for the more limited cross-reactivity of other monoclonal antibodies of the present invention which react significantly with peptidoglycans from only some bacterial species.
The importance of rapid detection of bacterial pathogens in clinical specimens (from the blood, tissues, and body spaces and cavities) is well-recognized. The standard method for determining the presence of bacteria comprises placement of the subject specimen in a medium which will support the growth of bacteria. Growth is detected by a variety of methods including direct observation, detection of nutrient utilization and detection of bacterial metabolic products. Since culture methods require a substantial time period for bacterial growth, implementation of appropriate antimicrobic therapy is delayed.
In normally sterile specimens such as blood and cerebrospinal fluid, the presence of bacteria indicates a potentially life threatening situation and dictates an immediate course of antobiotic therapy. The need for more rapid detection methods has given rise to a number of antibody-based detection methodologies. These methodologies employ antibodies from sera or from hybridoma cell lines which detect individual bacterial species or strains, but which lack the broad specificities of the monoclonal antibodies of the present invention. Appropriate diagnosis of bacterial infection or contamination depends on the ability to detect all bacteria.